The cell is the fundamental unit of life and no two cells are identical. For example, differences in genotype, phenotype and/or morphological property can contribute to cellular heterogeneity. Indeed, “seemingly identical” clonal populations of cells have been shown to display phenotypic differences among cells within the population. Cellular differences exist across all levels of life, ranging from bacterial cells to partially differentiated cells (for example, adult stem and progenitor cells) to highly differentiated mammalian cells (for example, immune cells). Differences in cellular state, function and responses can arise from a variety of mechanisms including different histories, different differentiation states, epigenetic variations, cell cycle effects, stochastic variations, differences in genomic sequence, gene expression, protein expression and differing cell interaction effects.
Conventional bulk cellular analyses, including measurements of expressed proteins or RNA, are performed by averaging very large numbers of cells, typically greater than 1000 cells per individual assay). This averaging of a cellular population masks the heterogeneity that exists within a cell population and obscures the underlying biological features of the individual cells within the population. There are many examples where such averaged measurements are inadequate. For example, measuring a cellular process in a cell population may be complicated by the responses of individual cells, which may be asynchronous, thus blurring the dynamics of the process. For example, the presence of dominant, yet phenotypically distinct subpopulations of cells can result in a population measurement that poorly reflects the internal states of the majority of cells in the population. See, e.g., Altschuler and Wu. (2010). Cell 141, pp. 559-563.
Existing methods for isolating populations of unique cell types are often limited in the purity of the population that is achievable. For example, enriched populations of primary multipotent stem cells rarely achieve better than 50% functional purity and are often well below 10% pure, so that the molecular signatures of these cells are obscured by large, and often overwhelming contamination from other cell types. Many cell types interact with each other, both through direct contact and through secreted factors, to promote survival, death, differentiation or some other function, and these interactions are difficult to isolate and study in a mixture comprising a large number of cells. Additionally, cells may have differences in their genomic sequences and/or cellular state that result in different levels or types of expressed mRNA or proteins. If analyzed in a bulk population, the particular cell with a unique cellular state or having the expressed mRNA or protein of interest, although of high value for industrial purposes, is very difficult or impossible to isolate from the population.
To overcome the deficiencies of bulk population cell analysis, single cell assay platforms have been developed. For example, microfluidic devices have been used to study single cells in the past (Lecault et al. (2012). Curr. Opin. Chem. Biol. 16, pp. 381-390). Ma et al. (Nat Med, 17, pp. 738-743 (2011)) applied a single cell barcode chip to simultaneously measure multiple cytokines (e.g., IL-10, TNF-β, IFN-γ) from human macrophages and cytotoxic T lymphocytes (CTLs) obtained from both healthy donors and a metastatic melanoma patient. Microfabricated chamber arrays have also been used to screen and select B cells secreting antigen-specific antibodies from both immunized humans and mice (Story et al. (2009). Proc. Natl. Acad. Sci. U.S.A. 105, pp. 17902-17907; Jin et al. (2009). Nat. Med. 15, pp. 1088-1092). In this approach, single B cells were arrayed on a surface containing tens of thousands of microfabricated wells (˜10-100 μm deep), where the well surfaces were functionalized with capture antibodies. After incubation of cells on the well surfaces for less than 3 hours, the surfaces were washed with fluorescently labeled antigen and scanned in order to identify antigen-specific B cells. These cells were then manually recovered from the arrays by a microcapillary in order to amplify, sequence, and clone the antibody-encoding genes from these cells.
Two-phase microfluidic devices have also been applied to the analysis of secreted proteins from single immune cells by encapsulating them in sub-nanoliter aqueous droplets separated by a stream of oil (Konry et al. (2011). Biosens. Bioelectron. 26, pp. 2702-2710). These droplets can be analyzed in a flow-through format similar to FACS, and thus provide an opportunity for ultra-high throughput detection of secreted proteins from single cells. Water-in-oil emulsions have also been used to study cellular paracrine signaling by co-encapsulating cells in microfluidic-generated agarose beads (Tumarkin et al. (2011). Integr. Biol. 3, pp. 653-662). Microfluidic droplet generation also has been used for drug screening and development by enabling viability analysis of encapsulated single cells exposed to different compositions (Brouzes et al. (2009). Proc. Natl. Acad. Sci. U.S.A. 106, pp. 14195-14200).
Antibodies are molecules naturally produced by the immune system of humans or animals to fight off infection and disease. This is achieved by the unique ability of the immune system to generate an immense diversity of antibodies, each with the ability to recognize and bind a specific target (e.g., protein, virus, bacteria). This unmatched specificity is also what makes antibodies extremely potent and low side-effect drugs with clinically approved therapies for a wide array of conditions including cancer, autoimmune disorders, inflammation, neurology, and infection. In comparison to conventional small molecule drugs, antibodies offer several advantages including superior pharmacokinetics, fewer side effects, improved tolerability, and much higher success rates in clinical trials (27% vs. 7% for small molecules). (Reichert (2009). Mabs 1, pp. 387-389.) It is for this reason that antibodies are also by far the fastest growing class of drugs, with a total global market that was $50B in 2012 and that is growing at a rate of 9% per year. (Nelson et al. (2010). Nat. Rev. Drug Disc. 9(10), pp. 767-774.)
The discovery of antibodies with optimal therapeutic properties, and in particular antibodies that target surface receptors, remains a serious bottleneck in drug development. In response to immunization, an animal can make millions of different monoclonal antibodies (mAbs). Each mAb is produced by a single cell called an antibody-secreting cell (ASC), and each ASC makes only one type of mAb. Accordingly, antibody analysis, for example, for drug discovery purposes lends itself to single cell analyses. However, even if an ASC is analyzed individually, and not within a bulk population of cells, because a single ASC generates only a minute amount of antibody, when analyzed in the volume of conventional assay formats, the antibody is too dilute, making it completely undetectable. Accordingly, new methods for studying individual ASCs and their secreted antibodies are needed. The present invention addresses this and other needs.